Depleting petroleum reserves, recurrent energy crises, increasing demand, and climate change have provided significant impetus in the search for sustainable technologies to replace petroleum as a source of fuels and chemical feedstocks. Long chain fatty acids and other derivatives are commercially attractive as fuel and chemical feedstocks because they can directly replace crude petroleum (as “bio-crude”), which is composed primarily of alkanes, alkenes, and aromatic hydrocarbons. In particular, cellulosic biomass is a preferred source of generating long chain fatty acids and other derivatives for use as fuel and chemical feedstocks, which are compatible with existing petroleum refining and distribution and can substitute for diesel, gasoline, jet fuel, and other derivatives of crude oil.
Currently, commercial and academic efforts are focused on bio-based petroleum replacement fuels made from microorganisms such as microalgae and that require aerobic microbial production. Algae bio-petroleum can appear as a very attractive option because fuel production occurs directly from sunlight and CO2. However, algal volumetric productivities are 100-fold lower than fermentative processes, requiring significantly higher biorefinery capital expenditures. See Liliana et al., Biotechnology and Bioengineering 102:100-12 (2009). In addition, lower capital algal options, such as open pond culturing, have many technical hurdles to clear before commercial deployment despite decades of research into the issue.
Other efforts are underway to produce fatty acid compounds from sugars and plant biomass, but all current methods require oxygen to be supplied during fermentation, and are not full consolidated bioprocessing (CBP) processes. Unlike traditional ethanol fermentations, aerobic biofuel synthesis routes feature product formation which is uncoupled from ATP generation and cell growth. Uncoupling of product formation from cell growth simplifies metabolic engineering and has allowed for rapid development of first generation biocatalysts. However, there is a price to be paid for aerobic production when the technology is scaled up to meet industrial needs. First, there are significant costs associated with scaling-up aerobic fermentations, such as, those due to the need for aeration and heat removal. In practice, these constraints limit the size of aerobic fermentors, with those used in anaerobic fuel ethanol production being an order of magnitude larger. Second, although maximum theoretical product yields from an aerobic process are only slightly lower than an anaerobic process, in practice it is extraordinarily difficult to approach this maximum since there is no biological incentive for microbes to reach high product yields.
To reach the best aerobic process hydrocarbon yields to date, researchers have resorted to high cell density fermentation, which resulted in product yields between 30-40% of the theoretical maximum. See Tsuruta et al., PLoS ONE 4:e4489 (2009); Whited et al., Industrial Biotechnology 6:152-163 (2010). While these yields may be quite acceptable for pharmaceutical or specialty chemical production, fuel biorefinery process models have shown that fermentation yields lower than 85% of theoretical result in unattractive process economics. However, an anaerobic, oxygen-free fermentation not only creates higher product yields, but also removes many significant scale-up problems associated with aerobic fermentation. Hydrocarbon fuel production also has process benefits compared to ethanol fuel production, such as a lower product recovery cost and a lower product toxicity to fermenting organisms. The latter could result in smaller fermentation volumes needed to reach equivalent productivities.
An anaerobic biocatalyst requires a higher degree of metabolic pathway integration to couple product formation with ATP generation, NAD(P)H regeneration, and cell growth. However, once these requirements are met, natural evolutionary forces can be harnessed to increase product yields and productivities, driving them towards theoretical maxima. See Burgard et al., Biotechnology and Bioengineering 84:647-57 (2003); Sauer, Advances in Biochemical Engineering/Biotechnology 73:129-69 (2001). Higher yields, combined with a lower-cost path for scale-up, make an anaerobic process a preferred option for developing microbes to produce fungible biofuels. The invention describes a method to produce long chain fatty acids and their derivatives in an organism or consortia of organisms in a CBP process that is anaerobic.
Integral to the process of producing any end product, including those that can be produced using the methods of the invention, is an adequate supply of metabolic substrates. Malonyl-CoA is such a key metabolic precursor for the biological synthesis of various bioproducts, including, but not limited to, fatty acid derived long chain hydrocarbon compounds such as fatty alcohols, fatty aldehydes, fatty acids, wax esters, and alkanes. However, the biosynthesis of malonyl-CoA is known to occur through only a few mechanisms in vivo—namely from acetyl-CoA, carbon dioxide, and ATP by acetyl-CoA carboxylase (acc, EC 6.4.1.2) or from malonate, CoA, and ATP by malonyl-CoA synthetase (matB) (An and Kim, Eur. J. Biochem. 257:395-402 (1998)). Yet, both of these mechanisms require the consumption of ATP to drive the reaction towards malonyl-CoA. In contrast, to produce fatty acid derived hydrocarbons, or any other bioproducts that use malonyl-CoA as a precursor, anaerobically at high yield, the route to malonyl-CoA should result in a net production of ATP. The invention describes recombinant microorganisms, pathways, and methods for producing desired end-products from malonyl-CoA precursors with a net production of ATP.